Transfection of GFP (Green Fluorescent Protein) into CHO Cells

 

Objectives

·        Transfect CHO cells with plasmid containing GFP.

·        Select for cells containing GFP.

 

Materials

Lipofectamine reagent

Plus reagent

RPMI 1640 medium

Antibiotic/antimycotic for RPMI 1640

TC-treated 6-well plates

Chinese Hamster Ovary (CHO) cells

DNA bacterial plasmids with GFP and antibiotic resistance, 1 mg/mL

G418 Antibiotic (Genecin), 100 mg/mL stock solution in dH₂O, sterile

1.5 mL eppindorf tubes

 

Safety

1.      Wear disposable latex gloves at all times

2.      Wear safety glasses at all times.

 

Prepare media (accomplished by T.A. or Instructor)

RPMI-A:  RPMI media without serum without antibiotic/antimycotic

RPMI-B:  RPMI media with 10% serum without antibiotic/antimycotic

RPMI-C:  RPMI media with 10% serum with 1% antibiotic/antimycotic

 

Experimental Summary

 

	Plate Cells	Transfect Cells	View Cells using F scope	Change/ Replenish Media
Day 1	X
Day 3		X
Day 5			X	X
Day 8			X	X
Day 10			X

 

Plate Cells (Day 1)

1.      Obtain a flask of CHO cells from the Instructor.  Cells will have been cultured by the Instructor in RPMI-C.

2.      Proceed through cell passaging protocol until step #15.  Use RPMI-B during passaging protocol.  Partners can share a flask of cells.

3.      After centrifugation, resuspend cells in 15 mL RPMI-B. 

4.      Determine the cell concentration using the Coulter Counter.

5.      Determine the cell dilution required to achieve a cell density of 50,000 cells/cm². Each well in a 6-well plate has a surface area of 10 cm² and holds 3 mL liquid. 

6.      Dilute the cells with RPMI-B to the calculated concentration. 

7.      Plate the cells at the calculated concentration and at 75%, 50%, and 25% of the desired concentration;  this should fill four of the six wells.  Also, select two other percentages of the calculated concentration (between 10% and 100%) or plan to replicate two of the percentages listed above.  Prepare dilutions as necessary and plate cells in the remaining two well.  Total volume in each well should be 3 mL. 

8.      Place cells in incubator for 2 days.

TARGET:  Cells at 50%-80% confluency on Day 3.  May need to adjust protocol and/or percentages to achieve these confluencies. 

 

Transfect Cells (Day 3)

1.      Check cells under microscope for confluency.  Record observations. 

2.      Label 3 1.5 mL eppindorf tubes.   

3.      Aliquot 1 mg of DNA plasmid into each eppindorf tube.  Inject liquid into bottom of tube.

4.      Add 6 mL Plus reagent to each eppindorf tube. Inject liquid into bottom of tube.

5.      Add 93 mL RPMI-A to each tube. Inject liquid into bottom of tube.  Mix by tapping bottom of tube on the hood surface.

6.      Let stand at room temperature for 15 min.

7.      Dilute 14 mL of Lipofectamine reagent in 336 mL of RPMI-A in separate eppindorf tube.  Mix by tapping bottom of tube on the hood surface.

8.      After 15 min incubation in Step 6., add 100 mL of diluted Lipofectamine reagent to each eppindorf tube.

9.      Let stand at room temperature for 15 min.

10.  While complexes are forming, remove cells from incubator.  Aspirate media from wells using glass Pasteur pipet. 

11.  Add 2 mL of RPMI-A to each well.  Aspirate media using glass Pasteur pipet immediately before step 13.

12.  After 15 min incubatrion in Step 9, add 800 mL of RPMI-A to each of the eppindorf tubes.  Mix gently using micropipette (bring liquid in and out of micropipette tip). 

13.  Using a micropipette, transfer the contents of each eppindorf tube into a well by dripping DNA/lipofectamine complexes onto the surface of each well. 

14.  Place cells in incubator for 3 hrs. 

15.  After incubation, aspirate liquid from wells using glass Pasteur pipet. 

16.  Add 3 mL RPMI-B to each well. 

17.  Add G418 antibiotic dropwise to a concentration of 800 mg/mL in each well.  G418 antibiotic must be kept sterile.  Rotate/shake plates gently to mix. 

18.  Replace wells in incubator.

 

View Cells (Day 5)

1.      Using a light microscope, estimate the confluency and fraction of attached and detached cells.  Look for dead cells.

2.      Using a fluorescence microscope, check for cells containing the GFP plasmid.  Record the percent of cells that contain GFP.  You will need to switch back and forth between the fluorescent and brightfield modes to make this estimate.

3.      Rinse each well two times with 1 mL PBS.  To rinse cells, do the following:

a)      Add 1 mL of rinse solution to each well using a micropipette.

b)      Swish lightly.

c)      Aspirate rinse solution using glass Pasteur pipet.

d)      Repeat, as given.

e)      Add 1 mL of PBS or RPMI-B to each well.

4.      Using a light microscope, estimate the confluency and fraction of attached and detached cells.  Look for dead cells.

5.   (10 pts)  Using a fluorescence microscope, check for cells containing the GFP plasmid.  Record the percent of cells that contain GFP.  You will need to switch back and forth between the fluorescent and brightfield modes to make this estimate. Estimate confluency of GFP-transfected cells.

 

Media Replenishment (Days 5 and 8)

1.      Refresh media nourishing cells in 6-well plates, as needed.

a)      Aspirate media from wells using glass Pasteur pipet. 

b)      Add 3 mL of RPMI-B to each well. 

c)      Add G418 antibiotic dropwise to a concentration of 800 mg/mL in each well.

d)      Rotate/shake plates gently to mix.

2.   Passage cells when confluency is near 100%.  Abbreviated protocol is given:

a)      Rinse cells with PBS.

b)      Add trypsin.  (Calculate the volume needed for each well of 6-well plate.)

c)      Place plate in incubator for 5 min.

d)      Add RPMI-B.

e)      Transfer to 15 mL centrifuge tube.  Add media up to 8 mL.

f)        Centrifuge.

g)      Resuspend in RPMI-B.

h)      Transfer fraction of cells to TC-treated petri dish (D = 100 mm).

i)        Add RPMI-B to total volume of 10 mL. 

j)        Add G418 antibiotic dropwise to a concentration of 800 mg/mL in each well.

k)      Rotate/shake plates gently to mix.

l)        Label petri dish and place in incubator

3.   Refresh media nourishing cells in petri dishes, as needed. Aspirate media from wells using glass Pasteur pipet.  Add 10 mL of RPMI-B to each plate.  Add G418 antibiotic dropwise to a concentration of 800 mg/mL in each well. Rotate/shake dishes gently to mix.

 

View Cells (Days 8 and 10)

1.      Using a light microscope, estimate the confluency and fraction of attached and detached cells.  Look for dead cells.

2.      Rinse each well two times with 1 mL PBS.  To rinse cells, do the following:

a)      Add 1 mL of rinse solution to each well using a micropipette.

b)      Swish lightly.

c)      Aspirate rinse solution using glass Pasteur pipet.

d)      Repeat, as given.

e)      Add 1 mL of PBS or RPMI-B to each well.

3.   Using a light microscope, estimate the confluency and fraction of attached and detached cells.  Look for dead cells.

4.   (10 pts per day)  Using a fluorescence microscope, check for cells containing the GFP plasmid.  Record the percent of cells that contain GFP.  You will need to switch back and forth between the fluorescent and brightfield modes to make this estimate.  Estimate confluency of GFP-transfected cells.  Are there any discernable patterns (rings, patches, etc.) of transfected cells?

 

 

Questions

1.      (10 pts)  Why is RPMI-B (media without antibiotic/antimycotic) used throughout most of experiment?

2.      (10 pts)  Do you see an increase in the percentage of cells that contain the plasmid containing GFP as a function of time?  Why or why not?

3.      (10 pts)  This experiment was conducted without any controls.  List the appropriate controls.

4.      (10 pts)  Does percent confluency increase or decrease after treatment with G418?  Postulate an explanation for the trend you observe.

5.      (10 pts)  Do you see any cells that appear dead?  If so, describe.

 

Observations and answers to Questions should be recorded in your laboratory notebook.  The point values are listed above.